A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein

Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrPC) into PrPSc, a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrPSc is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrPC may provide an opportunity to overcome these problems. PrPC ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrPC, and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrPC-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrPC-dependent synaptotoxicity of amyloid-beta (A beta) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrPC may produce a dual effect of blocking prion replication and inhibiting PrPC-mediated toxicity

Diagnostic and prognostic values of PBMC proteins in amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease for which there are no validated biomarkers. Previous exploratory studies have identified a panel of candidate protein biomarkers in peripheral blood mononuclear cells (PBMCs) that include peptidyl-prolyl cis-trans isomerase A (PPIA), heat shock cognate protein 71 kDa (HSC70), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) and TDP-43. It has also been found that PPIA plays a key role in the assembly and dynamics of ribonucleoprotein (RNP) complexes and interacts with TDP-43. Its absence accelerates disease progression in a SOD1 mouse model of ALS, and low levels of PPIA in PBMCs are associated with early-onset ALS. However, the diagnostic and prognostic values of PPIA and the other candidate protein biomarkers have not been established. We analyzed the PBMC proteins in a well-characterized cohort of ALS patients (n=93), healthy individuals (n=104) and disease controls (n=111). We used a highly controlled sample processing procedure that implies two-step differential detergent fractionation. We found that the levels of the selected PBMC proteins in the soluble and insoluble fraction, combined, have a high discriminatory power for distinguishing ALS from controls, with PPIA, hnRNPA2B1 and TDP-43 being the proteins most closely associated with ALS. We also found a shift toward increased protein partitioning in the insoluble fraction in ALS and this correlated with a worse disease phenotype. In particular, low PPIA soluble levels were associated with six months earlier death. In conclusion, PPIA is a disease modifier with prognostic potential. PBMC proteins indicative of alterations in protein and RNA homeostasis are promising biomarkers of ALS, for diagnosis, prognosis and patient stratification

Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis

Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133–134, 141, 152–153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.info:eu-repo/semantics/publishedVersio

Regulation of HuR structure and function by dihydrotanshinone-I

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity

Infectivity versus Seeding in Neurodegenerative Diseases Sharing a Prion-Like Mechanism

Prions are considered the best example to prove that the biological information can be transferred protein to protein through a conformational change. The term “prion-like” is used to describe molecular mechanisms that share similarities with the mammalian prion protein self-perpetuating aggregation and spreading characteristics. Since prions are presumably composed only of protein and are infectious, the more similar the mechanisms that occur in the different neurodegenerative diseases, the more these processes will resemble an infection. In vitro and in vivo experiments carried out during the last decade in different neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's diseases (PD), and amyotrophic lateral sclerosis (ALS) have shown a convergence toward a unique mechanism of misfolded protein propagation. In spite of the term “infection” that could be used to explain the mechanism governing the diversity of the pathological processes, other concepts as “seeding” or “de novo induction” are being used to describe the in vivo propagation and transmissibility of misfolded proteins. The current studies are demanding an extended definition of “disease-causing agents” to include those already accepted as well as other misfolded proteins. In this new scenario, “seeding” would be a type of mechanism by which an infectious agent can be transmitted but should not be used to define a whole “infection” process

Generation of a new infectious recombinant prion: a model to understand Gerstmann–Sträussler–Scheinker syndrome

Abstract Human transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders that include Kuru, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia. GSS is a genetically determined TSE caused by a range of mutations within the prion protein (PrP) gene. Several animal models, based on the expression of PrPs carrying mutations analogous to human heritable prion diseases, support that mutations might predispose PrP to spontaneously misfold. An adapted Protein Misfolding Cyclic Amplification methodology based on the use of human recombinant PrP (recPMCA) generated different self-propagating misfolded proteins spontaneously. These were characterized biochemically and structurally, and the one partially sharing some of the GSS PrPSc molecular features was inoculated into different animal models showing high infectivity. This constitutes an infectious recombinant prion which could be an invaluable model for understanding GSS. Moreover, this study proves the possibility to generate recombinant versions of other human prion diseases that could provide a further understanding on the molecular features of these devastating disorders

Unraveling the key to the resistance of canids to prion diseases

<div><p>One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different <i>in vitro</i> and <i>in vivo</i> models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the <i>Leporidae</i> and <i>Equidae</i> families. Here we undertake <i>in vitro</i> and <i>in vivo</i> approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis <i>in silico</i>, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids.</p></div

Dog PrP sequence alignments.

<p><b>A</b>. PrP amino acid alignment based on residues 91–230 (dog PrP numbering) from species with different experimental and/or natural susceptibilities to prion diseases. Classification criteria were based on the number of representative prion strains able to be transmitted and cause disease in the host. Identical amino acids are indicated by dots. Δ: number of different amino acids compared to dog PrP. Note the similarity of the amino acid sequence in the cat (highlighted in red), a species known to be susceptible to naturally acquired prion disease. <b>B</b>. Upper line, dog amino acid residues 91–230. The 6 amino acid differences compared to cat PrP sequence are highlighted by red squares. Boxes show each highlighted amino acid and representative species in which the particular amino acid is present also. Where prion susceptibility of the species has been proven the reference is provided. NP: susceptibility not proven. Positions 101 and 163 are polymorphic in dogs (Ser/Gly and Asp/Glu, respectively). Species codification and accession numbers: doPrP, dog PrP (FJ870767.1); hoPrP, horse PrP (ACG59277); raPrP, rabbit PrP (NP001075490); poPrP, porcine PrP (AAA92862.1); caPrP, cat PrP (ACB97675.1); huPrP, human PrP (NP001073592); mdPrP, mule deer PrP (AY330343.1); moPrP, mouse PrP (NP035300); boPrP, bovine PrP (ABR92636.1); ovPrP, ovine PrP (NP001009481.1); bvPrP, bank vole PrP (AAL57231.1); ahPrP, armenian hamster PrP (AAA37014); rcPrP, raccoon PrP (ACA50738.1); rsPrP, red squirrel PrP (AAN16491); coPrP, coyote PrP (AGA63673); gwPrP, grey wolf PrP (AGA63687); rfPrP, red fox PrP (ACA50742); huPrP; human PrP (U29185.1); niPrP, nilgai PrP (AAV30507); baPrP, California big-eared bat (AAN16503); anPrP, anteater PrP (AAN16516).</p